G protein-coupled receptor-in-paper, a versatile chromatographic platform to study receptor-drug interaction.

High-performance affinity chromatography is limited by its high cost and high pressure. Paper is made up of porous fiber networks and has the properties of low cost, ease of fabrication, and biodegradable. Due to these advantages, herein, we immobilized beta2-adrenoceptor (ß2-AR) onto the surface of the polytetrafluoroethylene membrane, a paper-based material, and constructed a G protein-coupled receptor (GPCR)-in-paper chromatographic platform. This platform was characterized by Fourier transform infrared spectroscopy, fluorescence analysis, X-ray photoelectron spectroscopy, and chromatographic studies. These morphological and elemental analysis showed that ß2-AR was successfully immobilized on the paper surface. The specific drugs have good retentions on the GPCR-in-paper chromatographic platform. The association constants of salbutamol, terbutaline and bambuterol to ß2-AR were calculated to be 2.02 × 104 M-1, 1.15 × 104 M-1, 1.75 × 104 M-1 by adsorption energy distribution, which were in good line with the values from frontal analysis, zonal elution and previous literatures. We demonstrated that the GPCR-in-paper platform was cost-effective, easy to be modified for protein immobilization, and applicable in the receptor-drug interaction analysis. We believe such a platform sheds new light on paper chromatography for receptor-drug interaction analysis and other applications.

A three-dimensional origami microfluidic device for paper chromatography: Application to quantification of Tartrazine and Indigo carmine in food samples.

Herein, we report three-dimensional paper chromatography (3D-PC) as a micro-chromatographic platform. The method was based on applying the origami microfluidic device for separation, coupled by colorimetric methods for simultaneous determination. The microfluidic device fabrication was a facile printing approach. Two azo food dyes, Tartrazine (E102) and Indigo carmine (E132), were selected as a model analyte, while carbonate-bicarbonate buffer was used as the mobile phase. Our micro-chromatographic device is associated with two big advantages including needing very small volume of mobile phase ( ~12 µL) and ultrafast separation time (~35 s). Under the optimal conditions, the method provided acceptable linear ranges of 0. 0 g L1-18.0 g L1 (R2 = 0.997) for E102 and 0.070 g L1-10.0 g L1 for E132 and the limits of detection (3σ/slope) were evaluated as 0.620 and 0.060 g L1, respectively. The proposed method was successfully applied in the separation and quantification of these dyes in commercial food products such as jelly, candy, and four kinds of drink samples without any sample preparation prior to analysis. The mean recovery values for the real sample analysis were in the range of 100.14%-102.38% for E132 and E102 respectively. The inter-device relative standard deviations were in the ranges of 1.5%-11.8%. In total, our chromatographic µPAD is small (1.0 cm × 1.0 cm × 0.5 cm), portable, inexpensive, no need of specialized user, requires low volumes of sample (0.5 µL), and can perform separation using 12 µL of aqueous mobile phase in very short time.

A paper-based analytical device coupled with electrochemical detection for the determination of dexamethasone and prednisolone in adulterated traditional medicines.

The adulteration of herbal medicines by dexamethasone or prednisolone is regarded as a serious problem in many communities. Herein, a novel platform for the separation and quantification of both target steroids in herbal medicines based on electrochemical paper-based analytical devices (ePADs) has been created. The ePAD was composed of Whatman SG81 chromatography paper, 3D-printed devices and a commercial screen-printed electrode. Whatman SG81 silica-coated paper was used for the separation of dexamethasone and prednisolone based on the difference in their partition coefficients during the flow of the mobile phase. The optimal mobile phase was composed of 60% ethyl acetate in cyclohexane and required 7 min for separation. The separated steroids on the paper were then quantified by electrochemical detection using differential pulse voltammetry, in which the 3D-printed devices facilitated the measurement. Analytical detection ranges of 10-500 µg mL-1 were obtained for both dexamethasone and prednisolone (r2 = 0.988 and 0.994, respectively). The limits of detection for dexamethasone and prednisolone were 3.59 and 11.98 µg mL-1, respectively, whereas the limits of quantification were 6.00 and 20.02 µg mL-1, respectively. The amounts of both target steroids derived from real herbal medicine samples determined by the proposed method were comparable to those obtained with assays using standard high-performance liquid chromatography. In addition, a simple evaporation step can be used to increase the concentration of the samples before analysis. These ePADs are simple, low-cost, rapid, and very promising for on-site quantitative detection.

Ampli: A Construction Set for Paperfluidic Systems.

The design and fabrication of reconfigurable, modular paperfluidics driven by a prefabricated reusable block library, asynchronous modular paperfluidic linear instrument-free (Ampli) block, are reported. The blocks are inspired by the plug-and-play modularity of electronic breadboards that lower prototyping barriers in circuit design. The resulting biochemical breadboard is a paperfluidic construction set that can be functionalized with chemical, biological, and electrical elements. Ampli blocks can form standard paperfluidic devices without any external instrumentation. Furthermore, their modular nature enhances fluidics in ways that fixed devices cannot. The blocks' ability to start, stop, modify, and reverse reaction flows, reagents, and rates in real time is demonstrated. These enhancements allow users to increase colorimetric signals, fine tune reaction times, and counter check multiplexed diagnostics for false positives or negatives. The modular construction demonstrates that field-ready, distributed fabrication of paper analytical systems can be standardized without requiring the "black box" of craft and technique inherent in paper-based systems. Ampli assembly and point-of-care redesign extends the usability of paper analytical systems and invites user-driven prototyping beyond the lab setting demonstrating "Design for Hack" in diagnostics.

Rapid multiplex nucleic acid amplification test developed using paper chromatography chip and azobenzene-modified oligonucleotides.

Although nucleic acid amplification test (NAT) is widely used for pathogen detection, rapid NAT systems that do not require special and expensive instruments must be developed in order to enable point of care (POC)-NATs, which contribute to early initiation of treatment. As a POC-NAT system, Kaneka DNA chromatography chip (KDCC), developed using DNA tag-bound primer through modified substance, was shown to be suitable for POC testing, due to the rapid detection time, simple procedures, and low manufacturing costs. However, owing to some modifications in primer, the detection performance and amplification speed were shown to be reduced when using KDCC, counteracting the increased speed of detection. To solve these issues and improve the speed of this NAT system, we investigated a better modification substance for KDCC. Here, azobenzene-modified primers were shown to have the highest amplification speed and detection performance in KDCC, of all modifications tested in this study, showing 10-100-fold lower detection limit but maintaining the same reaction time. Additionally, rapid herpes simplex virus detection system with azobenzene modified primers was developed. We believed that this breakthrough will contribute toward enabling greater utilization of POC-NATs for medical care, especially in developing countries and clinics.

Zinc oxide nanorods functionalized paper for protein preconcentration in biodiagnostics.

Distinguishing a specific biomarker from a biofluid sample containing a large variety of proteins often requires the selective preconcentration of that particular biomarker to a detectable level for analysis. Low-cost, paper-based device is an emerging opportunity in diagnostics. In the present study, we report a novel Zinc oxide nanorods functionalized paper platform for the preconcentration of Myoglobin, a cardiac biomarker. Zinc oxide nanorods were grown on a Whatman filter paper no. 1 via the standard hydrothermal route. The growth of Zinc oxide nanorods on paper was confirmed by a combination of techniques consisting of X-ray diffraction (XRD), X-ray photoelectron spectroscopy (XPS,) scanning electron microscopy (SEM), and energy dispersive spectroscopy (EDX) analysis. The Zinc oxide nanorods modified Whatman filter paper (ZnO-NRs/WFP) was further tested for use as a protein preconcentrator. Paper-based ELISA was performed for determination of pre-concentration of cardiac marker protein Myoglobin using the new ZnO-NRs/WFP platform. The ZnO-NRs/WFP could efficiently capture the biomarker even from a very dilute solution (Myoglobin < 50 nM). Our ELISA results show a threefold enhancement in protein capture with ZnO-NRs/WFP compared to unmodified Whatman filter paper, allowing accurate protein analysis and showing the diagnostic concept.

Impedance Analysis of Colloidal Gold Nanoparticles in Chromatography Paper for Quantitation of an Immunochromatographic Assay.

A detection method of gold nanoparticles in chromatography paper has been developed for a simple, cost-effective and reliable quantitation of immunochromatographic strip test. The time courses of the solution resistance in chromatography paper with the gold nanoparticles solution are electrochemically measured by chrono-impedimetry. The dependence of the solution resistance on the concentration of gold nanoparticles has been successfully observed. The main factor to increase the solution resistance may be obstruction of the ion transport due to the presence of gold nanoparticles. The existence of gold nanoparticles with 1.92 × 10(9) particles/mL in an indistinctly-colored chromatography paper is also identified by a solution resistance measurement. This indicates that the solution resistance assay has the potential to lower the detection limit of the conventional qualitative assay.

Ambient ionization based on mesoporous graphene coated paper for therapeutic drug monitoring.

Paper spray (PS), as a new ambient ionization method, has been applied for direct qualitative and quantitative analysis. The high sensitivity and minimum internal energy (low spray voltage) with optimized paper spray conditions is a significant request for real application in POCT. In this study, a simple and efficient ambient ionization method is developed by spraying from a mesoporous graphene foams (MGFs)-modified paper surface. The good electrical conductivity of MGFs results in obvious spray voltage decrease. Meanwhile, the MGFs-paper substrate has a well improvement in separation and elution efficiency ascribing to ultrahigh specific surface area and π-π electrostatic stacking property of graphene. In combination a commercial triple quadrupole mass spectrometer, the paper spray is successfully used for analysis of amphetamine in saliva. The linear dynamic ranges expand 10 fold in comparison with unmodified chromatography papers and the low limit of quantitation (LOQ) is as low as 1 pg/mL. A small sample volume (0.5 µL) could be analyzed immediately after spotting, without any pretreatment. The performance of this method was demonstrated for application in fast point-of-care mass spectrometry.

Visual detection technique for efficient screening and isolation of Salmonella based on a novel enrichment assay using chromatography membrane.

To detect Salmonella more efficiently and isolate strains more easily, a novel and simple detection method that uses an enrichment assay and two chromogenic reactions on a chromatography membrane was developed. Grade 3 chromatography paper is used as functionalized solid phase support (SPS), which contains specially optimized medium. One reaction for screening is based on the sulfate-reducing capacity of Salmonella. Hydrogen sulfide (H2S) generated by Salmonella reacts with ammonium ferric citrate to produce black colored ferrous sulfide. Another reaction is based on Salmonella C8 esterase that is unique for Enterobacteriaceae except Serratia and interacts with 4-methylumbelliferyl caprylate (MUCAP) to produce fluorescent umbelliferone, which is visible under ultraviolet light. A very low detection limit (10(1) CFU ml(-1)) for Salmonella was achieved on the background of 10(5) CFU ml(-1) Escherichia coli. More importantly, testing with more than 1,000 anal samples indicated that our method has a high positive detection rate and is relatively low cost, compared with the traditional culture-based method. It took only 1 day for the preliminary screening and 2 days to efficiently isolate the Salmonella cells, indicating that the new assay is specific, rapid, and simple for Salmonella detection. In contrast to the traditional culture-based method, this method can be easily used to screen and isolate targeted strains with the naked eye. The results of quantitative and comparative experiments showed that the visual detection technique is an efficient alternative method for the screening of Salmonella spp. in many applications of large-sized samples related to public health surveillance.

Alternative chromatographic system for the quality control of lipophilic technetium-99m radiopharmaceuticals such as [(99m)Tc(MIBI)6].

Knowledge of the radiochemical purity of radiopharmaceuticals is mandatory and can be evaluated by several methods and techniques. Planar chromatography is the technique normally employed in nuclear medicine since it is simple, rapid and usually of low cost. There is no standard system for the chromatographic technique, but price, separation efficiency and short time for execution must be considered. We have studied an alternative system using common chromatographic stationary phase and alcohol or alcohol:chloroform mixtures as the mobile phase, using the lipophilic radiopharmaceutical [(99m)Tc(MIBI)6]⁺ as a model. Whatman 1 modified phase paper and absolute ethanol, Whatman 1 paper and methanol:chloroform (25:75), Whatman 3MM paper and ethanol:chloroform (25:75), and the more expensive ITLC-SG and 1-propanol:chloroform (10:90) were suitable systems for the direct determination of radiochemical purity of [(99m)Tc(MIBI)6]⁺ since impurities such as (99m)Tc-reduced-hydrolyzed (RH), (99m)TcO(4)(-) and [(99m)Tc(cysteine)2]⁻ complex were completely separated from the radiopharmaceutical, which moved toward the front of chromatographic systems while impurities were retained at the origin. The time required for analysis was 4 to 15 min, which is appropriate for nuclear medicine routines.

Alternative chromatographic system for the quality control of lipophilic technetium-99m radiopharmaceuticals such as [99mTc(MIBI)6]+

Knowledge of the radiochemical purity of radiopharmaceuticals is mandatory and can be evaluated by several methods and techniques. Planar chromatography is the technique normally employed in nuclear medicine since it is simple, rapid and usually of low cost. There is no standard system for the chromatographic technique, but price, separation efficiency and short time for execution must be considered. We have studied an alternative system using common chromatographic stationary phase and alcohol or alcohol:chloroform mixtures as the mobile phase, using the lipophilic radiopharmaceutical [99mTc(MIBI)6]+ as a model. Whatman 1 modified phase paper and absolute ethanol, Whatman 1 paper and methanol:chloroform (25:75), Whatman 3MM paper and ethanol:chloroform (25:75), and the more expensive ITLC-SG and 1-propanol:chloroform (10:90) were suitable systems for the direct determination of radiochemical purity of [99mTc(MIBI)6]+ since impurities such as99mTc-reduced-hydrolyzed (RH),99mTcO4- and [99mTc(cysteine)2]-complex were completely separated from the radiopharmaceutical, which moved toward the front of chromatographic systems while impurities were retained at the origin. The time required for analysis was 4 to 15 min, which is appropriate for nuclear medicine routines.

A simple method for patterning poly(dimethylsiloxane) barriers in paper using contact-printing with low-cost rubber stamps.

This paper presents a simple and low-cost method for patterning poly(dimethylsiloxane) (PDMS) barriers in porous support such as paper for the construction of flexible microfluidic paper-based analytical devices (µPADs). The fabrication method consisted of contact-printing a solution of PDMS and hexane (10:1.5 w/w) onto chromatographic paper using custom-designed rubber stamps containing the patterns of µPADs. After penetrating the paper (∼30 s), the PDMS is cured to form hydrophobic barriers. Under optimized conditions, hydrophobic barriers and hydrophilic channels with dimensions down to 949±88 µm and 771±90 µm (n=5), respectively, were obtained. This resolution is well-suitable for most applications in analytical chemistry. Chemical compatibility studies revealed that the PDMS barriers were able to contain some organic solvents, including acetonitrile and methanol, and aqueous solutions of some surfactants. This find is particularly interesting given that acetonitrile and methanol are the most used solvents in chromatographic separations, non-aqueous capillary electrophoresis and electroanalysis, as well as aqueous solutions of surfactants are suitable mediums for cell lyses assays. The utility of the technique was evaluated in the fabrication of paper-based electrochemical devices (PEDs) with pencil-drawn electrodes for experiments in static cyclic voltammetry and flow injection analysis (FIA) with amperometric detection, in both aqueous and organic mediums.

The detection of blood group phenotypes using paper diagnostics.

BACKGROUND AND OBJECTIVES: Paper biodiagnostics for blood typing are novel, cheap, fast and easy to use. Agglutinated red blood cells cannot travel through the porous structure of paper, indicating a positive antibody-antigen interaction has occurred. Conversely, non-agglutinated blood can disperse and wick through the paper structure with the ease to indicate a negative result. This principle has been demonstrated to detect blood group phenotypes: ABO and RhD. However, typing for red blood cell antigens such as Rh, Kell, Duffy and Kidd has not yet been explored on paper. MATERIALS AND METHODS: Two paper testing methods - an elution and a direct flow-through method - were investigated to detect red blood cell antigens excluding the ABO system and RhD. Antigens explored include the following: C, c, E, e, K, k, Fy(a), Fy(b), Jk(a), Jk(b), M, N, S and s, P1, Le(a) and Le(b). The variables tested include the following: reaction time and reagent concentration. The importance of antibody type/structure for successful agglutination on paper was confirmed. RESULTS: Some blood group phenotypes showed less agglutination due to weaker antibody-antigen interactions. Most blood groups with antibodies available as IgM, such as C, c, E, e, K and k, and Jk(a) and Jk(b), and P1, were successful using both methods. However, other blood groups, especially those with antibodies only available as polyclonal antibodies, were unsuccessful and require further scrutiny. CONCLUSION: Paper can be used as an alternative blood grouping diagnostic tool for selected blood group phenotypes.

Two-dimensional paper chromatography-based fluorescent immunosensor for detecting acute myocardial infarction markers.

A novel washing scheme following antigen-antibody reactions with analyte was used during construction of a fluorescent immunosensor to resolve the background problem in the lateral flow assay with human serum. An immuno-membrane strip was devised to simultaneously measure cardiac troponin I (cTnI), creatinine kinase-MB isoform (CK-MB), and myoglobin to diagnose acute myocardial infarction. This strip was then installed within a cartridge containing a built-in washing solution tank, which was used to supply the solution across the signal generation pad of the strip after the immune reactions. Such cross-flow washing was initiated by onset-signaling from the internal control and began to run automatically upon sample addition. Under optimal conditions, the immunosensor displayed a stably suppressed background baseline, enabling us to attain a low detection limit for cTnI (0.05 ng/mL) as well as favorable reproducibility for repetitive measurements (relative standard deviation <10%). No interference was observed among the different complex formations at the respective analyte sites, and no artifacts were caused by sample matrices. We tested the performance relationship with the Pathfast reference system for positive serum samples (36 for cTnI, 58 for CK-MB, and 17 for myoglobin), and the correlation coefficients were >0.98. This result suggests that the new immunosensor system based on two-dimensional chromatography can be used for clinical testing.

Evanescent field: a potential light-tool for theranostics application.

A noninvasive or minimally invasive optical approach for theranostics, which would reinforce diagnosis, treatment, and preferably guidance simultaneously, is considered to be major challenge in biomedical instrument design. In the present work, we have developed an evanescent field-based fiber optic strategy for the potential theranostics application in hyperbilirubinemia, an increased concentration of bilirubin in the blood and is a potential cause of permanent brain damage or even death in newborn babies. Potential problem of bilirubin deposition on the hydroxylated fiber surface at physiological pH (7.4), that masks the sensing efficacy and extraction of information of the pigment level, has also been addressed. Removal of bilirubin in a blood-phantom (hemoglobin and human serum albumin) solution from an enhanced level of 77 µM/l (human jaundice >50 µM/l) to ~30 µM/l (normal level ~25 µM/l in human) using our strategy has been successfully demonstrated. In a model experiment using chromatography paper as a mimic of biological membrane, we have shown efficient degradation of the bilirubin under continuous monitoring for guidance of immediate/future course of action.

Assessment of the within- and between-lot variability of Whatman™ FTA(®) DMPK and 903(®) DBS papers and their suitability for the quantitative bioanalysis of small molecules.

BACKGROUND: To ensure that PK data generated from DBS samples are of the highest quality, it is important that the paper substrate is uniform and does not unduly contribute to variability. This study investigated any within and between lot variations for four cellulose paper types: Whatman™ FTA(®) DMPK-A, -B and -C, and 903(®) (GE Healthcare, Buckinghamshire, UK). The substrates were tested to demonstrate manufacturing reproducibility (thickness, weight, chemical coating concentration) and its effect on the size of the DBS produced, and the quantitative data derived from the bioanalysis of human DBS samples containing six compounds of varying physicochemical properties. RESULTS & DISCUSSION: Within and between lot variations in paper thickness, mass and chemical coating concentration were within acceptable manufacturing limits. No variation in the spot size or bioanalytical data was observed. CONCLUSION: Bioanalytical results obtained for DBS samples containing a number of analytes spanning a range of chemical space are not affected by the lot used or by the location within a lot.

Three-dimensional paper-based microfluidic device for assays of protein and glucose in urine.

The first step in curing a disease is being able to detect the disease effectively. Paper-based microfluidic devices are biodegradable and can make diagnosing diseases cost-effective and easy in almost all environments. We created a three-dimesnional (3D) paper device using wax printing fabrication technique and basic principles of origami. This design allows for a versatile fabrication technique over previously reported patterning of SU-8 photoresist on chromatography paper by employing a readily available wax printer. The design also utilizes multiple colorimetric assays that can accommodate one or more analytes including urine, blood, and saliva. In this case to demonstrate the functionality of the 3D paper-based microfluidic system, a urinalysis of protein and glucose assays is conducted. The amounts of glucose and protein introduced to the device are found to be proportional to the color change of each assay. This color change was quantified by use of Adobe Photoshop. Urine samples from participants with no pre-existing health conditions and one person with diabetes were collected and compared against synthetic urine samples with predetermined glucose and protein levels. Utilizing this method, we were able to confirm that both protein and glucose levels were in fact within healthy ranges for healthy participants. For the participant with diabetes, glucose was found to be above the healthy range while the protein level was in the healthy range.

Validation of an alternative radiochemical purity method for [99mTc]pentetate ([99mTc]DTPA).

[(99m)Tc]pentetate ([(99m)Tc]DTPA) is the most commonly used radiopharmaceutical renography agent. The aim of this work was to validate an alternative method for assessing [(99m)Tc]DTPA radiochemical purity (RCP), according to the ICH Q2(R1) guidance: "Validation of Analytical Procedures". The proposed method is composed of two chromatographic systems. System A is a miniaturized system of thin layer chromatography (TLC) silica gel impregnated aluminum strips as stationary phase (SP) and distilled water as mobile phase (MP). System B consists of Whatman 1 paper strips as SP and methyl ethyl ketone as MP. Results indicate that the proposed RCP method has been validated, as it is specific, precise, accurate, linear and robust. Therefore, it can be used as an alternative method for RCP quality control purposes and as stability indicator as well.

Paper analytical devices for fast field screening of beta lactam antibiotics and antituberculosis pharmaceuticals.

Reports of low-quality pharmaceuticals have been on the rise in the past decade, with the greatest prevalence of substandard medicines in developing countries, where lapses in manufacturing quality control or breaches in the supply chain allow substandard medicines to reach the marketplace. Here, we describe inexpensive test cards for fast field screening of pharmaceutical dosage forms containing beta lactam antibiotics or combinations of the four first-line antituberculosis (TB) drugs. The devices detect the active pharmaceutical ingredients (APIs) ampicillin, amoxicillin, rifampicin, isoniazid, ethambutol, and pyrazinamide and also screen for substitute pharmaceuticals, such as acetaminophen and chloroquine that may be found in counterfeit pharmaceuticals. The tests can detect binders and fillers such as chalk, talc, and starch not revealed by traditional chromatographic methods. These paper devices contain 12 lanes, separated by hydrophobic barriers, with different reagents deposited in the lanes. The user rubs some of the solid pharmaceutical across the lanes and dips the edge of the paper into water. As water climbs up the lanes by capillary action, it triggers a library of different chemical tests and a timer to indicate when the tests are completed. The reactions in each lane generate colors to form a "color bar code" which can be analyzed visually by comparison with standard outcomes. Although quantification of the APIs is poor compared with conventional analytical methods, the sensitivity and selectivity for the analytes is high enough to pick out suspicious formulations containing no API or a substitute API as well as formulations containing APIs that have been "cut" with inactive ingredients.

Methods for measuring Class I membrane-bound hyaluronan synthase activity.

Detecting and quantifying hyaluronan (HA) made by Class I HA synthase (HAS) and determining the level of activity of these membrane-bound enzymes is critical in studies to understand the normal biology of HA and how changes in HAS activity and HA levels or size are important in inflammatory and other diseases, tumorigenesis, and metastasis. Unlike the products made by the vast majority of glycosyltransferases, HA products are more complicated since they are made as a heterogeneous population of sizes spanning a broad mass range. Three radioactive and nonradioactive assay methods are described that can give the amount of HA made with or without information about the distribution of product sizes.